Cardiovascular disease (CVD) is the number one cause of morbidity and mortality worldwide. An estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths. Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke. More remarkably, low- and middle-income countries are disproportionally affected, driving the need for regenerative therapies in lieu of chronic drug treatment regimens and such regenerative therapies must be offered in formats eliminating the need for high cost laboratory infrastructure or extensive multi-hour usage of vascular catheter labs. Over 80% of CVD deaths take place in low- and middle-income countries and occur almost equally in men and women. In the progression of CVDs, plaque lesions develop in arteries that result in the narrowing of vessels, and in severe cases they break open and create a blockage of blood flow (ischemia) to a vital part of the heart, brain or limb. Such ischemia may be reversed if treated within a short period of time by reperfusion therapy and further by the infusion of adult tissue derived stem cells with or without the presence of extracellular factors. Despite significant advances in medical therapy and revascularization strategies, the prognosis of certain patients with Acute Myocardial Infarction (AMI), Chronic Heart Failure (CHF), Critical Limb Ischemia (CLI) and Ischemic Brain Injury (Stroke) remains dismal without the introduction of early biological repair intervention.
Along with reperfusion, adjuvant stem cell therapy has been shown to be potentially efficacious in the repair and regeneration of damaged tissue of heart, brain and limbs from ischemic injury. These stem/progenitor cells can be isolated from different sources and one such source is bone-marrow. The autologous, bone-marrow derived in one case, peripheral blood derived in the second case, and adipose derived in the third, adult stem/progenitor cells circumvent the ethical and legal issues related to embryonic stem cells. Further, it also terminates the risk of transmitting diseases and immune rejection. The regenerative potential of autologous stem cells, specifically adipose or peripheral blood, and most specifically bone marrow derived cellular product is highly influenced by the aspiration, processing, and delivery technique employed. Anticoagulant plays a crucial role in the overall efficacy of regenerative cell therapy, and extended exposure to anticoagulant has been shown to have a negative impact on the “stemness” of the cells. The use of an anticoagulant in the aspiration syringe and processing devices keeps the bone-marrow in a non-coagulated state, which allows proper stratification, isolation and infusion of stem/progenitor cells to treat clinical conditions. Anticoagulant also inhibits to varying degrees the formation of microthrombae, which if injected into the patient can cause adverse events. It is also understood that the addition of any chemical entity in the presence of proteins or cells (“Biologicals”) may have an effect on the structure or function of the Biologicals. Therefore, the anticoagulant used, the concentration used, and the time of cell exposure to the anticoagulant individually and potentially, cumulatively, plays a crucial role in the overall outcome of the efficacy and safety of stem cell therapy. The ideal stem cell compatible anticoagulant must be in the optimal concentration ratio balance to keep the cell therapy concentrate or infusate in the anti-coagulated state for a defined period of time to complete the interventional procedure and also reduce or eliminate the intravascular formation of thrombae while minimizing the risk of peri-procedural bleeding and modulation of stem cells.
Heparins are the most commonly used anticoagulant agents for bone-marrow aspiration. It has, however, been reported that heparin can interfere with the mobilization and homing mechanism(s) of stem and/or progenitor cells thereby impairing the functional therapeutic capacity of these cells. Moreover, heparins are associated with high rates of peri-procedural bleeding, which may be related to their inability to bind to clot-bound thrombin. Still further, heparins can get inactivated in-vivo by binding to platelet factor-4 (PF-4). Certain individuals expressing antibody to heparin-PF-4 complex can experience Heparin-Induced-Thrombocytopenia (HIT) on exposure to heparins.
The anti-coagulated bone marrow aspirate is in a heterogenous mixture containing Hematopoietic Stem Cells (“HSCs”), Mesenchymal Stem Cells (MSCs), Endothelial Progenitor Cells (“EPCs), CXCR4 positive cells, White Blood Cells (“WBCs”), Red Blood Cells (“RBCs”), platelets (“PLTs”), plasma with serum proteins and metabolites (“PLASMA”), and fat (“FAT”) and is unsuitable for delivery in the raw aspirate form into a patient's vascular system. Furthermore, to properly define the therapeutic cell mixture constituency for reproducible therapeutic efficacy patient-to-patient, and to prepare a safe but dose specific volume of the therapeutic cell concentrate, the therapeutically effective cells (HSC's, MSC's, WBC's, EPC's and CXCR4 positive cells) must be purified, isolated or captured from the ineffective and undesired cells and fat in a short period of time to render a concentrated therapeutic dose of cells and factors at the point-of-care.
The purification of cells, and specifically WBCs containing the desired HSC's, EPC's, MSC's and other stem/progenitors cells, has historically been accomplished by placing the cells in a containment device and applying standard centrifugal stratification under a gravity force using either chemical density based methods or in automated chemical free systems. The cells of the bone marrow coming from the red marrow and stroma will stratify to their respective density under specific gravity force (“g force”) in a defined period of time, and thus create layers of cells in the containment device from bottom to top wherein the RBCs (most dense) are on the bottom, followed by the nucleated RBC's and VSELs, the WBC's, PLTs, PLASMA, and FAT. Both of the methods simply harvest the buffy coat as typically defined as the cellular layer above the RBC/nucleated RBC fraction and below the PLT fraction.
The ideal cell purification process involves a closed minimally manipulated procedure for rapidly stratifying and isolating the desired HSCs, EPCs, MSCs plus other stem/progenitor cells, which may also be referred to as stem cells or MNCs or progenitor cells or bone marrow concentrate enriched in progenitor cells (BMCEPC) throughout this document, away from the undesired mature RBCs and topping up the desired cell fraction with PLASMA to the proper volume and viscosity for safe and effective delivery into the vascular system via a stem cell friendly device such as an over the wire balloon catheter, or an intra-organ delivery cannula.
The delivery of stem cells via a catheter procedure is an ideal way of infusing cells proximally to the afflicted organ. However, trans-catheter passage of cells can potentially have a negative impact on the desired cells in several ways. First, the cells undergo varying shear stresses depending on their proximity to the lumen wall, the velocity of the injection, the radius of the lumen, and the viscosity of the fluid, and second since the lumen is a polymeric substrate its potential to impact cells and cell membranes, which come in contact through activation, binding or shedding can be important.
The transcatheter delivery of stem cells into the vascular system is desirably achieved by introduction of an intravascular catheter in the peripheral vasculature, and inducing transient ischemia in the targeted organ proximally or distally to the catheter tip by inflating an intravascular balloon to slow or stop blood flow for a pre-determined time. Such ischemia improves the cellular homing of the stem and progenitor cells to the ischemic tissue, and increases the opportunity for the cells initiating one or more processes including but not limited to tissue integration, cellular fusion, cellular differentiation, and/or paracrine factor release. Ischemia is best induced by inflating a compliant or non-compliant balloon to reduce or stop blood flow while taking extra care to minimize any endothelium wall damage from the balloon pressurization.
Thus, there is a need for an improved method and composition for the aspiration, processing, and delivery of bone marrow cells, such that the bone-marrow derived stem cells provide a safe and efficient adjuvant therapy for re-vascularization and or organ repair in ischemic disorders.